Protein synthesis is a highly conserved process in all kingdoms of life that can be broken down into four distinct phases: initiation, elongation, termination and ribosome recycling. The broad goals of this research program are to use biochemical and genomic approaches to shed light on the common and distinctive molecular features of translation elongation, termination, and recycling in bacteria and eukaryotes, and their control. Here we are particularly focused on one aspect of translational control in which ribosomal stalling triggers a cellular response leading to mRNA decay, targeted proteolysis, and ribosome recycling. In particular, we focus on a highly conserved stalling motif, the poly-basic peptide sequence that is of particular relevance in eukaryotic cells where alternative polyadenylation site usage commonly leads to non-stop mRNAs. We will use in vitro biochemistry and in vivo ribosome profiling to look at the molecular mechanics of this biologically important and conserved process. More specifically, we propose (1) to use reporters and our previously established in vitro reconstituted translation systems (with E. coli and S. cerevisiae components) to ask a series of questions about how poly-basic sequences disrupt ribosome function during elongation, (2) to use these same reporters and in vitro biochemistry to define how different extra-ribosomal factors engage the ribosome and impact elongation, termination and recycling and (3) to use recently developed ribosome profiling approaches to define the biologically relevant in vivo targets, their molecular features, and the factors that respond in the cell to resolve the crisis. We anticipate that the synergy of these approaches will be powerful in defining biologically relevant mechanism.

Public Health Relevance

mRNA surveillance pathways (including non-stop decay, NSD) are critical in regulating gene expression throughout the three kingdoms of life. Of key relevance to this proposal, non-stop mRNAs are commonly generated (as much as 5-10% of transcripts) in eukaryotic cells when cryptic poly(A) sites are utilized; such events have been implicated as relevant for a number of diseases [Klauer, 2012 #100]. Mechanistic understanding of NSD will be required for the development of effective therapies to treat such diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM059425-16
Application #
9133391
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1999-05-01
Project End
2019-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
16
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205
Schuller, Anthony P; Zinshteyn, Boris; Enam, Syed Usman et al. (2018) Directed hydroxyl radical probing reveals Upf1 binding to the 80S ribosomal E site rRNA at the L1 stalk. Nucleic Acids Res 46:2060-2073
Schuller, Anthony P; Green, Rachel (2018) Roadblocks and resolutions in eukaryotic translation. Nat Rev Mol Cell Biol 19:526-541
Dever, Thomas E; Dinman, Jonathan D; Green, Rachel (2018) Translation Elongation and Recoding in Eukaryotes. Cold Spring Harb Perspect Biol 10:
Guydosh, Nicholas R; Green, Rachel (2017) Translation of poly(A) tails leads to precise mRNA cleavage. RNA 23:749-761
McClary, Brandon; Zinshteyn, Boris; Meyer, Mélanie et al. (2017) Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A. Cell Chem Biol 24:605-613.e5
Guydosh, Nicholas R; Kimmig, Philipp; Walter, Peter et al. (2017) Regulated Ire1-dependent mRNA decay requires no-go mRNA degradation to maintain endoplasmic reticulum homeostasis in S. pombe. Elife 6:
Schuller, Anthony P; Green, Rachel (2017) The ABC(E1)s of Ribosome Recycling and Reinitiation. Mol Cell 66:578-580
Schuller, Anthony P; Wu, Colin Chih-Chien; Dever, Thomas E et al. (2017) eIF5A Functions Globally in Translation Elongation and Termination. Mol Cell 66:194-205.e5
Zinshteyn, Boris; Green, Rachel (2016) When stop makes sense. Science 354:1106
Radhakrishnan, Aditya; Chen, Ying-Hsin; Martin, Sophie et al. (2016) The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality. Cell 167:122-132.e9

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